The highest Duffy (FYX) allele frequency ever reported for Scottish population: A cross‐sectional study

Abstract Background and Aim The Duffy (FY) blood group system has six known antigens among which the Fya and Fyb are known as major antigens. Fyx phenotype forms as a result of two point mutations in the FYB allele leading to instability of Duffy protein and so reduction of Duffy antigen expression in the cells. This study aimed to investigate the FYX allele frequency in the Scottish population. Methods The Duffy blood group system was serologically and molecularly investigated in 222 samples collected from donors of Aberdeen Regional Blood Transfusion Center (BTC). The haemagglutination and BeadChip microarray chemistry methods were used for phenotyping and genotyping. Confirmatory tests were also used to check the discrepant results. Results In this study, the frequency of Duffy blood group phenotypes including Fya+, Fya+b+, and Fyb+ were 17.57%, 42.79%, and 39.64%, respectively. Furthermore, the frequency of FYA/FYA, FYA/FYB, and FYB/FYB genotypes was estimated to be 14.41%, 45.95%, and 39.64%, respectively, using the Bioarray method. In the present study, based on Duffy DNA sequencing results, 12 samples (5.41%) had just one FYX allele. Conclusion The frequency of the FYX allele in this study was estimated to be 0.0270% which is more than the results reported so far.

anti-Fy b in the serum of a woman who had been pregnant three times, Fy b was discovered. 6 The Fy x phenotype is a weak form of Fy b that was reported later by two other groups. 7,8 The FYX allele has a frequency of 2%-3.5% in Caucasian people while it is not seen in Blacks. 9 FYA and FYB alleles differ by a single G to A nucleotide substitution at position 125 in exon 2. 10 The molecular basis of the Fy (a-b − ) phenotype is related to the mutation in the GATA promoter region of the FYB (−67, T to C). Disturbance at the binding site for the GATA-1 erythroid transcription factor can be considered a result of this nonsense mutation. 9 Moreover, a similar mutation is observed in the GATA-1 promoter region of the FYA. 11 The highest frequency for Fy b was observed when a group studied the frequency of Fy a and Fy b in Blacks using the polymerase chain reaction with restriction fragment length polymorphism (PCR-RFLP) method. 12 Owing to the high incidence of discrepancies between Fy b phenotyping results versus genotyping outcomes on the collected samples from Scottish blood donors through the PCR-RFLP method, Murphy suggested that the frequency of Fy x in Caucasians was more than the previously reported 0.015%. 13,14 To determine the pregnancies at risk for HDFN, Hessner et al. used a validated PCR-ASP assay for prenatal genotyping in 1999. The method utilized was able to determine the FYA, FYB, null FYA, and null FYB alleles. 15 One year later, the two missense mutations related to FYX were defined using the PCR with sequence-specific primers (PCR-SSP) method in 300 samples. 16 Schmid and co-workers reported a frequency of 1% for Fy x haplotypes in 54 samples of African American blood donors in 2011. 17 In 2015, Silvia Manfroi and co-workers used the BLOODchip ® IDcor + technique to determine the FYX/FY null and FYA/FY null genotypes in a Caucasian thalassemia family from Sardinia and investigated the FYX and FY null alleles frequency in Caucasian and black people. 9 In the present study, the prevalence of the FYX allele was measured in a Scottish population using high-sensitivity molecular assays.

| MATERIALS AND METHODS
To study the Duffy blood group serologically and molecularly, blood samples were taken from 222 blood donors. Forty-seven of the donors (male = 19, female = 28) were healthy and partially known reference panel donors of Aberdeen Regional Blood Transfusion Center (BTC), and the other blood samples were taken from active, healthy platelet donors (n = 175, male = 126, female = 49) who consented to participate in the study. The collection of blood samples began after the project was approved by the Grampian Local and Regional Ethics Committee (LREC) and the Scottish National Blood Transfusion Services (SNBTS) Clinical Governance committee. The purified genomic DNA (gDNA) of buffy coat from whole blood EDTA samples was genotyped by BeadChip microarray chemistry, that is, BioArray HEA BeadChip™ and their red blood cells were used for phenotyping. The commercial kits, READY GENE KKD PCR-SSP, in-house DNA-PCR SSP assay, and serological methods were used for re-genotyping and re-phenotyping due to the existence of discrepant results. The mentioned methods are overviewed in the section below.

| BeadChip microarray
Microarray-based technologies are known as powerful techniques to study gene expression patterns on a genome-wide scale. [18][19][20] Recently, the human erythrocyte antigen (HEA) BeadChip array of bioarray solutions, which has been used in the present study to investigate the samples, has become more popular as microarray chemistry. In our study, the HEA24 and HEA28 BeadChip kits were used for genotyping of donor samples. The following five steps are necessary to use these kits; multiplex PCR amplification of target markers on the gDNA, cleaning up of the PCR products using ExoSAP-IT, generating singlestrand DNA (ssDNA) products using Lambda Exonuclease, hybridization/and elongation of the ssDNA to the specific probes on the BeadChips and finally reading the images using a Microarray AIS400 system (Bioarray Solutions) and analyzing the data online. 21 The results of allele frequency, allele detection rates (ADR), corrected allele detection rate (CADR), and concordance rates (CoR) were calculated based on the following formula, respectively: where n, AA, nʹ, AB, and N denote the number of detected homozygous, homozygous, the number of detected heterozygous, heterozygous, and total sample number, respectively.
where N is the total number of the tested samples, nIC is the total number of samples that showed intermediate call (IC) and nʹLS is the total number of samples with low signal.
This formula was used when the warning message for a blood group was for only one allele and the s nd allele gave a correct result.

| Serological test
All the blood samples collected for the BioArray study (BA study) including platelets and reference panels were phenotyped serologically for Fy a and Fy b . The blood group phenotyping was performed based on the technical recommendations from suppliers for each monoclonal antibody/antiserum. All the samples with a discrepancy of serological results for Fy b were re-phenotyped using two different polyclonal anti-Fy b to check and confirm the donors' phenotype results.

| In-house PCR-SSP
To produce large amounts of a specific DNA fragment of defined length and sequence from a small amount of a template, the PCR can be used as an in-vitro method. 21,22 In this study, we used optimized and validated PCR-SSP.

| Commercial PCR-SSP kits
These kits are produced by different companies and are expected to perform more robustly than in-house protocols since they usually pass different stages of the validation process and quality controls before being introduced to the market. In this study, the commercial PCR-SSP kit (READY GENE KKD PCR-SSP Kit) was used as an additional method for confirmation and verification of some achieved results.

| RESULTS
The serological blood group phenotyping and the HEA BeadChip™ genotyping results are as follows:

| Haemagglutination blood group phenotyping results
The haemagglutination blood group phenotyping results are presented in Table 1.

| Microarray blood group genotyping results
The results of the blood group genotyping, the determined allele frequencies, overall ADR for the Duffy blood group as well as CoR between predicted genotypes from haemagglutination and genotyping results of microarray assay are shown in Table 1.
In the present study, 0 of 222 samples showed IC or LS. ADR and CADR were 100%.
As it is shown in Table 1, genotype discrepancies versus serological phenotyping outcomes occurred in seven donors possessing Fy b (n = 7; BA048, BA078, BA115, BA141, BA155, BA162, and RP35, which are based on the sample ID). As mentioned before, the discrepant samples were re-phenotyped and re-genotyped and this will be discussed in the following section.
T A B L E 1 Summary of Duffy blood group phenotypes and actual genotype results from hemagglutination and microarray typing of 222 platelet and reference panel donors from Aberdeen Regional SNBTS.

| Re-phenotyping of discrepant samples
The re-phenotype results with different antisera were in good agreement with the original serological results that were obtained using antisera from Alba Bioscience with slight differences in their grades of hemagglutination (±1 grade variation of reaction at the most).

| Re-genotyping of discrepant samples
To validate the results obtained by HEA BeadChip assays for the FYB discrepant samples, the Inno-TRAIN KKD PCR-SSP kit was used as one of the confirmatory tests. A summary of the results is presented in Table 2.
Moreover, the in-house PCR-SSP has been also used as a confirmatory test and its results are in good agreement with the results of HEA BeadChip assays and commercial conventional PCR-SSP kits.

| DNA sequencing of discordant samples
The  Table 3.
To investigate the GATA mutation at the promoter region, the positions of nucleotide 125 G > A (Nt 797), which encoded the FYA/ T A B L E 2 Summary of the results from KKD PCR-SSP typing for seven discrepant and a control patient sample with SCD (MA).   Table 3).

| DISCUSSION
In the present study, in 12 of 222 Scottish donors (5.41%), one FYX allele was discovered (see Table 3). The frequency of the FYX allele was estimated to be 0.0270%. This is in agreement with a study conducted by Murphy et al., 14  FYX -associated mutations were in the other allele. Since these two samples were Fy b+ve by the usual serologic typing, missense mutation at 298 position alone does not seem adequate to change the Fy b expression at the significant level and prevent the other FYB allele from being read. The existence of this mutation has been reported in 33% of Caucasians. 32 In the present study, in contrast to the 265 C > T mutation, the 298 G > A polymorphism did not lead to instability of Fy b protein antigen and reduction of antigen expression in erythrocytes.

| CONCLUSION
This study aimed to investigate the FYX allele frequency in the Scottish population. The frequency of the FYX allele was estimated to be 0.0270% which is, to the best of our knowledge, the highest frequency reported so far in the Scottish population. However, further studies with larger sample sizes are recommended to evaluate the FYX allele frequency more precisely.

CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

TRANSPARENCY STATEMENT
The lead author Ali Mohammad Varzi affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.